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目的观察健脾补土组方对新生SD鼠神经元体外培养低氧/复氧后的凋亡情况以及PI3K、Akt、Caspase-3表达的影响,探讨其减少神经元凋亡的作用机制。方法原代分离培养新生SD鼠神经元,随机分为正常血清对照组、低氧模型组、神经生长因子组、健脾补土组。将标本低氧诱导24 h,再复氧培养4 h进行造模。用流式细胞术检测神经元凋亡情况,RTq PCR法检测PI3K、Akt、caspase-3 mRNA的表达,免疫细胞化学法和Western blot法检测PI3K、Akt、caspase-3蛋白的表达。结果与正常对照组相比,低氧模型组神经元凋亡率显著增加,PI3K、Akt mRNA与蛋白表达显著减少,Caspase-3 mRNA与蛋白表达显著增加;与低氧模型组比较,神经生长因子组、健脾补土组神经元凋亡率均显著减少,PI3K、Akt mRNA及蛋白表达显著增强,Caspase-3 mRNA及蛋白表达显著较少(P<0.01);与神经生长因子组比较,健脾补土组神经元凋亡率显著减少,PI3K、Akt mRNA及蛋白表达显著增加,Caspase-3 mRNA及蛋白表达显著降低。结论健脾补土组方能减少脑缺血后神经元的凋亡,其作用机制可能与其通过上调PI3K和Akt的表达水平,最终抑制在细胞凋亡中起关键作用的因子Caspase-3的表达有关。
Objective To observe the effects of invigorating the spleen and invigorating herbs on the apoptosis of neurons in vitro of hypoxia / reoxygenation and the expression of PI3K, Akt and Caspase-3 in cultured neonatal SD rat neurons in vitro and to explore the mechanism of action of decreasing the neuronal apoptosis. Methods Primary cultured neonatal SD rat neurons were randomly divided into normal serum control group, hypoxia model group, nerve growth factor group and Jianpi Tumo group. The specimens were induced by hypoxia for 24 h and then reoxygenated for 4 h for modeling. The apoptosis of neurons was detected by flow cytometry. The expressions of PI3K, Akt and caspase-3 mRNA were detected by RT-PCR. The expressions of PI3K, Akt and caspase-3 were detected by immunocytochemistry and Western blot. Results Compared with the normal control group, the apoptosis rate of neurons in hypoxia model group was significantly increased, the expressions of PI3K, Akt mRNA and protein were significantly decreased and the expression of Caspase-3 mRNA and protein were significantly increased. Compared with hypoxia model group, the expression of nerve growth factor (P <0.01). Compared with the nerve growth factor group, the apoptosis rate of neurons in the group of invigorating the spleen and invigorating the spleen was significantly decreased, the expression of PI3K, Akt mRNA and protein were significantly increased, and the expression of Caspase-3 mRNA and protein was significantly decreased The apoptotic rate of neurons in spleen-remnant group was significantly decreased, the expressions of PI3K, Akt mRNA and protein were significantly increased, and the expressions of Caspase-3 mRNA and protein were significantly decreased. CONCLUSION: Jianpi Tumo Decoction can reduce the neuronal apoptosis after cerebral ischemia, and its mechanism may be related to it by inhibiting the expression of PI3K and Akt and ultimately inhibiting the expression of Caspase-3, a key factor in apoptosis related.